From Quantitative Microscopy
New horizons in biology and medicine are opened by technologies that allow the investigation of molecular mechanisms which regulate cell/tissue function and disfunction. We (ENI, UU, UCAM, MRC) aim to develop novel integrated detection systems for quantitative detection of emission spectra, fluorescence lifetime and polarization. New techniques and technologies have to be user-friendly and cost-effective in order to allow non-specialist biomedical laboratories to take advantage of biophysical imaging. Also, cost-effective systems will permit the implementation of new tools for drug-discovery and diagnostics.
On the right: a CCD/CMOS camera for wide-field FLIM.
Innovating Quantitative Microscopy
In the following sections, the three most innovative developments are described:
- the first confocal time-resolved spectropolarimeter
- the first unsupervised high throughput FLIM
- the first single-shot parallel fast wide-field FLIM
INNOVATING TECHNOLOGY TO PUSH BOUNDARIES OF BIOLOGY
Hyper-Dimensional Imaging Microscopy
- imaging spectropolarimetry:
- time-resolved spectropolarimetry:
We will present images aquired with the spectropolarimeter at the Biophysical Society Meeting. This section will be completed soon after.
Unsupervised FLIM for high throughput imaging of biochemical events
Proteomics and Cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Foerster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular sub-populations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatio-temporal cellular responses to chemical and physical stimuli. We demonstrated that the high content offered by these techniques can be combined with the high-throughput levels offered by automation of a fluorescence lifetime imaging microscope setup, capable of unsupervised operation and image analysis. Systems and software dedicated to Image Cytometry for Analysis and Sorting represent important emerging tools for the field of p roteomics, interactomics and cellomics.
Fast and cost-effective FLIM
FD-FLIM systems were limited by the use of expensive technologies and specialized instrumentation, by limited spatial resolution and acquisition throughput and limited capability to resolve heterogeneous systems. We developed methods of analysis and novel technologies to overcome those limitations and to foster the engineering of the new generation of sensing technologies.
- fast and cost-effective system: Solid state technologies for sensing (CMOS and CCD) and sample excitation (LED and laser diodes) were combined in the first cost-effective (~12k€ + microscope) prototype of a FD imaging system capable of full field imaging with a single exposure. This technology may replace in the near future the obsolete multi-channel plates used in intensified camera providing fast and efficient FLIM systems. * Innovative FLI-CAM
- multi-exponential decays: Typical FD-FLIM wide field systems provided limited capabilities in evaluating heterogeneous (non single exponential) fluorescence decays on a pixel-by-pixel basis. We developed new techniques which complemented global analysis methods for the estimation of lifetime heterogeneity. More recently, we further developed global analysis methods and pixel-by-pixel estimators to resolve bi-exponential decays.
- signal-to-noise ratio: Acquisition throughput and spatial resolution are limited by the physical limits of the sensing techniques and also by noise. Also for the best use of novel solid state technologies and integration of the inexpensive light emitting diodes and CCD/CMOS lock-in imagers we analysed the noise performances of FLIM systems to determine the best excitation/detection schemes (short, 1-2ns, light pulses at 40-80Mhz repetition rate for typical fluorophores) and to understand the physical limitations of lifetime sensing.
Pages in category "Technology"
The following 6 pages are in this category, out of 6 total.
- Fluorescence Lifetime Imaging Microscopy
- Fluorescence lifetime imaging microscopy: quality assessment and standards
- Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics